6 research outputs found
NMR chemical shift perturbations in Bm-Aspin due to pepsin interactions.
<p>Perturbations in the chemical shift position of the residues Y215 (a), I214 (b), and A213 (c) respectively in Bm-Aspin upon addition of increasing concentrations of pepsin. Ratios of Bm-Aspin to pepsin are: 1∶0 (red), 1∶0.1 (cyan), 1∶0.5 (green), and 1.1 (yellow).</p
Kinetics of aspartic protease inhibition by Bm-Aspin.
<p>Lineweaver-Burk Plots showing the variation (1/V with that of 1/S) of competitive inhibition of pepsin (A) and cathepsin-E (C), non-competitive for renin (B) and mixed inhibition for cathepsin-D (D) respectively. Assays were carried out in triplicates, with the fixed quantity of proteases (5 mM) and varying concentrations of Bm-Aspin (0 mM, 1 mM, 2.5 mM and 5 mM) The inhibition constants were determined using Graphpad Prism 2.0 (San Diego, CA).</p
Bm-Aspin <sup>15</sup>N HSQC spectra at varying concentrations of SDS.
<p>Comparison of Bm-Aspin <sup>15</sup>N HSQC spectra in the presence of varying concentrations of SDS; (<b>i</b>) 50 mM SDS, (ii) 100 mM SDS, (iii) 150 mM SDS, and (iv) 200 mM SDS. The inset box indicates the well resolved glycine peaks for comparison to identify the optimum solvent conditions for a well behaved NMR spectrum.</p
Chemical shift perturbations upon addition of human aspartic proteases.
<p>(A) NMR Chemical shift Perturbations in Bm-Aspin due to different protease interactions at their saturated conditions. Chemical shift perturbations of the residues Y215 (i), I214 (ii), and A213 (iii) respectively, upon the addition of human aspartic proteases at their saturation levels. Free Bm-Aspin (Magenta), Bm-Aspin+Cathepsin-D (Yellow), Bm-Aspin+Cathepsin-E (Green), Bm-Aspin+Renin (Red), Bm-Aspin+Pepsin (Grey). (B) Comparison of Bm-Aspin chemical shift perturbation upon addition of human aspartic proteases. The bar diagram indicating the radial shift (calculated for each of the affected residues, by combining both the chemical shifts of <sup>1</sup>H and <sup>15</sup>N, using the equation: Radial shift displacement (Δδ) = {(H<sub>f</sub>−H<sub>b</sub>) <sup>2</sup>+[(N<sub>f</sub>−N<sub>b</sub>)/6] <sup>2</sup>} <sup>1/2</sup>. A scaling factor of 6 was used to normalize the differences in the <sup>1</sup>H and <sup>15</sup>N spectral widths. H<sub>f</sub>, H<sub>b</sub>, N<sub>f</sub>, and N<sub>b</sub> are the chemical shifts of each residue's amide <sup>1</sup>H and <sup>15</sup>N in the <b>free</b> (Bm-Aspin alone) and <b>bound</b> (Bm-Aspin+protease complex) states, respectively) in ppm, observed due to NMR chemical shift perturbations in Bm-Aspin with the addition of proteases, (Bm-Aspin+Pepsin in blue, Bm-Aspin+Renin in red, Bm-Aspin+Cathepsin-E in green, Bm-Aspin+Cathepsin-D in magenta), observed for the following 10 residues: G16, G22, G82, G169, G190, A192, A204, A213, I214, and Y215.</p
NMR screening on Bm-Aspin with different detergents.
<p><sup>15</sup>N HSQC spectra of Bm-Aspin at pH 7.0 with the addition of: (i) No detergent (ii) 0.5 M Urea and 1% glycerol (iii) 1% <i>n</i>-octyl-β-D-glucoside (OG), (iv) 100 mM n-Dodecyl β-D-Maltopyranoside (DDM) (v) 1% triton X-100, and (vi) 100 mM SDS.</p
Sequential residue connectivities using triple resonance NMR strip plot.
<p>Plot showing the strips of four triple resonance NMR spectra of Bm-Aspin in the following order: HNCOCA, HNCA, CBCACONH and HNCACB. The sequential <sup>13</sup>Cα connectivities for the residues' stretch T189-V194 are indicated by the continuous line drawn between the adjacent Cα.</p